CN101941967A - Salt of 13a-(S) deoxidized tylophorinine, preparation method, pharmaceutical composition and application thereof - Google Patents
Salt of 13a-(S) deoxidized tylophorinine, preparation method, pharmaceutical composition and application thereof Download PDFInfo
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Abstract
The invention relates to a salt of (+)-13a-(S)-deoxidized tylophorinine, a preparation method of the compounds, a pharmaceutical composition comprising the compounds and application of the compounds to preparing medicines for preventing and/or treating cancers and/or inflammatory diseases.
Description
Technical field
The present invention relates to the salt of a kind of 13a-(S) deoxidation tylophorinine, the preparation method of this compounds, the pharmaceutical composition and this compounds that contain them prevent and/or treat application in the medicine of cancer and/or inflammatory disease in preparation.
Background technology
Phenanthroindolizididerivative pyridine alkaloid mainly is distributed in the Asclepiadaceae Tylophora plant, has multiple pharmacologically active, and wherein antitumor action and anti-inflammatory action attract people's attention.Find that in the antitumor screening of American National cancer research institute (NCI) this Alkaloid has significant effect for 60 kinds of tumor cell lines, half growth-inhibiting dosage (GI
50) 10
-8M level, and, good selectivity is arranged such as melanoma and lung carcinoma cell for malignant tumour, effective for the resistance cancerous cell line, and do not have cross resistance with other anticarcinogen.
(+)-13a-(S)-deoxidation tylophorinine (has been applied for the compound patent, application number 200610076298.X) be to be located away from phenanthroindolizididerivative pyridine alkaloid in three fens pellets of Asclepiadaceae Tylophora plant, have very strong anti-tumor activity, experiment in vitro shows the IC of this compound to Ketr3, HCT-8, A549, BGC-803, Bel-7402, B16BL6, KB, CaSE-17, HL-60 tumor cell line
50Between 0.1～0.3 μ M, illustrate (+)-13a-that (S)-deoxidation tylophorinine has extracorporeal anti-tumor function.In vivo test shows that (+)-13a-(S)-deoxidation tylophorinine has the obvious suppression effect for the growth of rat liver cancer H22 and Mice Bearing Lewis Lung Cancer, shows that it still has better antitumor activity in vivo.Preliminary toxicity test shows that this toxicity of compound is lower, does not show liver, renal toxicity.
To studies show that of the mechanism of action of this compounds anti-tumor activity, it is for DNA and the RNA and proteinic synthetic all influential (the Bioorg Med Chem Lett.2006 of cell, 16:4300-4304.), further molecular pharmacology studies show that, the mechanism of action of the various antitumour drugs of its mechanism of action and clinical application is completely different, this compounds has optionally potent restraining effect for the NF-κ B signal path that influences the rna transcription process, and this action intensity and mutual corresponding [the MolCancer Ther.2006 of the intensity of its cytotoxic activity, 5 (10): 2484-2493.], but concrete action target spot also research among.
The anti-inflammatory action of this compounds is also relevant with its inhibition NF-κ B signal path, and closely related [the Mol Pharm.2006 of effect of this effect and the MEKK1 of its blocking-up NF-κ B signal path upstream, 69 (3): 749-758.], but concrete action target spot is still unknown.
(+)-13a-(S)-deoxidation tylophorinine fat-soluble higher, water insoluble, but water-soluble significantly raising will be found behind its salify, find that by anti tumor activity in vitro screening and pharmacodynamic experiment its anti-tumor activity is maintained, the pharmacokinetic property of the salt by measuring (+)-13a-(S)-deoxidation tylophorinine is found, four kinds of salt being tested average retention time (MRT) in the mouse body obviously prolongs, bioavailability (AUC) is compared quite with (+)-13a-(S)-deoxidation tylophorinine or is slightly high, and wherein the bioavailability of maleate is apparently higher than the prototype medicine.
Summary of the invention
The technical problem that will solve of the present invention is to provide acceptable hydrate or prodrug on general formula (I) compound and the pharmacodynamics thereof.
The another technical problem that the present invention will solve is to provide the method for preparing acceptable hydrate on general formula (I) compound and the pharmacodynamics thereof or prodrug.
The another technical problem that the present invention will solve is to provide a kind of pharmaceutical composition, and it comprises acceptable hydrate or prodrug and pharmaceutical carrier and/or vehicle at least one general formula (I) compound and the pharmacodynamics thereof.
The technical problem again that the present invention will solve is to provide acceptable hydrate on general formula (I) compound and the pharmacodynamics thereof or prodrug to be used for preventing and/or treating the application of cancer and/or inflammation medicine in preparation.
For solving the problems of the technologies described above, the technical solution used in the present invention is:
The present invention is about the compound suc as formula (I):
Wherein, HX represents organic acid or mineral acid;
When HX represented mineral acid, it can comprise, but be not limited to hydrochloric acid, sulfuric acid, phosphoric acid, Hydrogen bromide;
When HX represented organic acid, it can comprise, but be not limited to tartrate, Citric Acid, toxilic acid, lactic acid, Whitfield's ointment, oxysuccinic acid, phenylformic acid, hexanodioic acid, fumaric acid, succsinic acid.
According to the present invention, the compound shown in the preferred general formula (I) comprises, but is not limited to the compound shown in the general formula (IA):
Wherein, HX is preferably from tartrate, Citric Acid, toxilic acid, lactic acid.
According to the present invention, preferred compound include but not limited to following compound:
In order to finish the present invention's purpose, the present invention takes following technical scheme:
Concrete operations comprise the steps:
(+)-13a-(the S)-deoxidation tylophorinine (CAT-1) and the corresponding sour HX of equivalent are suspended in the dehydrated alcohol, and 50 ℃ of following stirring reactions 30 minutes, it is clear and bright that reaction solution becomes, and reaction solution put be chilled to room temperature, obtains corresponding salt solid behind the evaporate to dryness.
The solid that obtains ethyl alcohol recrystallization purifying.
The preparation of (+)-13a-(S)-deoxidation tylophorinine is with reference to patent " dextrorotation deoxidation tylophorinine among the present invention, its method for making and its pharmaceutical composition and purposes " (application number 200610076298.X) and patent " 13a-(S) deoxidation tylophorinine derivative, its method for making and pharmaceutical composition and purposes " (application number 200910079163.2).By with corresponding sour salify, its activity is maintained, pharmacokinetic property is optimized.
The invention still further relates to a kind of pharmaceutical composition that contains medicine effective dose as described compound of general formula I and pharmaceutically acceptable carrier.
The invention still further relates to and contain as the The compounds of this invention of active ingredient and the pharmaceutical composition of conventional medicine vehicle or assistant agent.Usually pharmaceutical composition of the present invention contains the The compounds of this invention of 0.1～95 weight %.The general content of The compounds of this invention is 0.1～100mg in unit dosage form, and preferred unit dosage form contains 4～50mg.
The pharmaceutical composition of The compounds of this invention can be according to method preparation well known in the art.When being used for this purpose, if desired, The compounds of this invention and one or more solids or liquid medicine vehicle and/or assistant agent can be combined, make and can be used as suitable administration form or the dosage form that people's medicine or veterinary drug use.
The compounds of this invention or contain its pharmaceutical composition can the unit dosage form administration, route of administration can be enteron aisle or non-enteron aisle, as oral, muscle, subcutaneous, nasal cavity, oral mucosa, skin, peritonaeum or rectum etc.
The compounds of this invention or the route of administration that contains its pharmaceutical composition can be drug administration by injection.Injection comprises intravenous injection, intramuscular injection, subcutaneous injection, intradermal injection and acupoint injection therapy etc.
Form of administration can be liquid dosage form, solid dosage.As liquid dosage form can be true solution class, colloidal type, particulate formulations, emulsion dosage form, mixed suspension form.Other formulations are tablet, capsule, dripping pill, aerosol, pill, pulvis, solution, suspensoid, emulsion, granule, suppository, lyophilized injectable powder etc. for example.
The compounds of this invention can be made ordinary preparation, also can be sustained release preparation, controlled release preparation, targeting preparation and various particulate delivery system.
For example, can be extensive use of various carrier well known in the art for the unit form of administration is made tablet.Example about carrier is, for example thinner and absorption agent are as starch, dextrin, calcium sulfate, lactose, N.F,USP MANNITOL, sucrose, sodium-chlor, glucose, urea, lime carbonate, white bole, Microcrystalline Cellulose, pure aluminium silicate etc.; Wetting agent and tackiness agent are as water, glycerine, polyoxyethylene glycol, ethanol, propyl alcohol, starch slurry, dextrin, syrup, honey, glucose solution, mucialga of arabic gummy, gelatine size, Xylo-Mucine, lac, methylcellulose gum, potassiumphosphate, polyvinylpyrrolidone etc.; Disintegrating agent, for example dry starch, alginates, agar powder, laminaran, sodium bicarbonate and Citric Acid, lime carbonate, polyoxyethylene sorbitol fatty acid ester, sodium laurylsulfonate, methylcellulose gum, ethyl cellulose etc.; Disintegration inhibitor, for example sucrose, Tristearoylglycerol, theobroma oil, hydrogenation wet goods; Absorption enhancer, for example quaternary ammonium salt, sodium lauryl sulphate etc.; Lubricant, for example talcum powder, silicon-dioxide, W-Gum, stearate, boric acid, whiteruss, polyoxyethylene glycol etc.Tablet further can also be made coating tablet, for example sugar coated tablet, thin membrane coated tablet, ECT, or double-layer tablets and multilayer tablet.
For example, can be extensive use of various carrier well known in the art for pill is made in the administration unit.Example about carrier is, for example thinner and absorption agent are as glucose, lactose, starch, theobroma oil, hydrogenated vegetable oil, polyvinylpyrrolidone, single stearic acid glycerine lipoprotein, kaolin, talcum powder etc.; Tackiness agent is as gum arabic, tragacanth gum, gelatin, ethanol, honey, liquid sugar, rice paste or batter etc.; Disintegrating agent is as agar powder, dry starch, alginates, sodium laurylsulfonate, methylcellulose gum, ethyl cellulose etc.
For example for capsule is made in the administration unit, the effective constituent The compounds of this invention is mixed with above-mentioned various carriers, and the mixture that will obtain thus places hard gelatine capsule or soft capsule.Also the effective constituent The compounds of this invention can be made microcapsule, be suspended in and form suspensoid in the aqueous medium, in the hard capsule of also can packing into or make injection and use.
For example, The compounds of this invention is made injection preparation, as solution, suspensoid solution, emulsion, lyophilized injectable powder, this preparation can be moisture or non-water, can contain acceptable carrier, thinner, tackiness agent, lubricant, sanitas, tensio-active agent or dispersion agent on a kind of and/or multiple pharmacodynamics.Can be selected from water, ethanol, polyoxyethylene glycol, 1 as thinner, the isooctadecanol of ammediol, ethoxylation, the isooctadecanol of polyoxyization, polyoxyethylene sorbitol fat, fat acid esters etc.In addition, ooze injection liquid, can in injection preparation, add proper amount of sodium chloride, glucose or glycerine, in addition, can also add conventional solubility promoter, buffer reagent, pH regulator agent etc. in order to prepare etc.These auxiliary materials are that this area is commonly used.
In addition, as needs, also can in pharmaceutical preparation, add tinting material, sanitas, spices, correctives, sweeting agent or other material.
For reaching the medication purpose, strengthen result of treatment, medicine of the present invention or pharmaceutical composition can be with any known medication administrations.
The dosage of The compounds of this invention pharmaceutical composition depends on many factors, for example to prevent or treat the character and the severity of disease, the sex of patient or animal, age, body weight, personality and individual reaction, route of administration, administration number of times, therapeutic purpose, therefore therapeutic dose of the present invention can have large-scale variation.In general, the using dosage of Chinese materia medica composition of the present invention is well known to a person skilled in the art.Can be according to the actual drug quantity that is contained in the preparation last in the The compounds of this invention composition, in addition suitable adjustment to reach the requirement of its treatment significant quantity, is finished prevention of the present invention or therapeutic purpose.The suitable dose scope of the every day of The compounds of this invention: the consumption of compound of the present invention is 0.001～100mg/Kg body weight, is preferably 0.1～60mg/Kg body weight, and more preferably 1～30mg/Kg body weight most preferably is 2～15mg/Kg body weight.Be 10～500mg The compounds of this invention every day that adult patient is taken, and is preferably 20～100mg, can once take or divide and take for 2～3 times; The dosage of children taking is preferably 10～20mg/kg body weight according to every kg body weight 5～30mg.Above-mentioned dosage can the single dose form or be divided into several, for example two, three or four dosage form administrations, this is subject to administration doctor's the clinical experience and the dosage regimen of treatment means.Compound of the present invention or composition can be taken separately, or merge use with other treatment medicine or symptomatic drugs.
The invention still further relates to the application of compound of the present invention in the medicine of preparation treatment cancer and/or inflammatory disease, particularly the application in cancers such as human colon carcinoma, people's cancer of the stomach, human ovarian cancer, cervical cancer, liver cancer, lung cancer, carcinoma of the pancreas, lymphatic cancer and neural friendship matter knurl.
Useful technique effect of the present invention:
Aromizing takes place and produces impurity in (+)-13a-(S)-deoxidation tylophorinine fat-soluble higher, water insoluble easily under illumination or air, by its salify is found later on, and water-soluble increasing substantially, and stability increases.
Carry out external activity screening and pharmacodynamic experiment by salt to (+)-13a-(S)-deoxidation tylophorinine, find the quite active of its anti-tumor activity and prototype compound (+)-13a-(S)-deoxidation tylophorinine, illustrate (+)-13a-that (S)-deoxidation tylophorinine salify later stage anti-tumor activity is maintained.
Carry out back blood plasma pharmacokinetic by tartrate, maleate and citrate to (+)-13a-(S)-deoxidation tylophorinine, find that experiment mice is very fast to the absorption of these salt, peak time is short, and these salt average retention time (MRT) in the mouse body is compared obvious prolongation with (+)-13a-(S)-deoxidation tylophorinine; And the bioavailability of these salt (AUC) and (+)-13a-(S)-deoxidation tylophorinine are quite or improve, will be apparently higher than the prototype medicine such as the bioavailability of maleate; Behind mouse oral (+)-13a-(S)-deoxidation tylophorinine tartrate, maleate and the citrate, find that by measuring the content of prototype medicine in animal brain the medicament contg of tartrate, 3 time points of maleate all is higher than the prototype medicine.After this explanation (+)-13a-(S)-deoxidation tylophorinine salify, its pharmacokinetic property has obtained optimization.
Description of drawings
The influence of Fig. 1 (+)-13a-(S)-deoxidation tylophorinine and salt pair H22 growth of xenografted thereof
The influence of Fig. 2 (+)-13a-(S)-deoxidation tylophorinine and salt pair lotus knurl KM mouse body weight thereof
The influence of Fig. 3 (+)-13a-(S)-deoxidation tylophorinine and salt pair H22 growth of xenografted thereof
Fig. 4 (+)-13a-(S)-deoxidation tylophorinine mice plasma typical curve
Curve when Fig. 5 mouse oral (+)-13a-(S)-three kinds of salt of deoxidation tylophorinine and prototype medicine (6mg/kg) blood plasma medicine
Fig. 6 mouse oral (+)-13a-(S)-deoxidation tylophorinine tartrate (6mg/kg) hindbrain is organized medicament contg
Fig. 7 mouse oral (+)-13a-(S)-deoxidation tylophorinine maleate (6mg/kg) hindbrain is organized medicament contg
Fig. 8 mouse oral (+)-13a-(S)-deoxidation tylophorinine citrate (6mg/kg) hindbrain is organized medicament contg
Embodiment
Used initial compounds in the embodiment of the invention, can be according to the ordinary method of this area and/or method well known to those skilled in the art preparation, and can be by the preparation of following preparation example for example.
The following examples are used for further specifying the present invention, but this and do not mean that any limitation of the invention.
The english abbreviation explanation:
CTX: endoxan
LC/MS/MS: liquid phase mass spectrometry
DMSO: dimethyl sulfoxide (DMSO)
MRT (0-∞): average retention time
AUC (0-∞): bioavailability
T1/2z: transformation period
Tmax: peak time
Cmax: peak concentration
Preparation example: the preparation of (+)-13a-(S)-deoxidation tylophorinine
With reference to patent " dextrorotation deoxidation tylophorinine, its method for making and its pharmaceutical composition and purposes " (application number 200610076298.X) and patent " 13a-(S) deoxidation tylophorinine derivative, its method for making and pharmaceutical composition and purposes " (application number 200910079163.2), the product specific rotation is
(c=0.25, CHCl
3102 ° of)=+, ee=99.1%[chirality AD-H post; Moving phase: Virahol: normal hexane (15: 85), 0.1% triethylamine; λ=254nm; T (major)=18.79min, t (minor)=26.09min], ESI-MS:364.2[M+H]
+,
1H-NMR (400MHz, CDCl
3): 7.92 (1H, d, J=9.2Hz, H-1), 7.21 (1H, dd, J=9.2Hz, 2Hz, H-2), 7.88 (1H, d, J=2Hz, H-4), 7.90 (1H, s, H-5), 7.12 (1H, s, H-8), 4.00 (3H, s, MeO), 4.04 (3H, s, MeO), 4.09 (3H, s, MeO), 4.62 (1H, d, J=14.8Hz, H-9), 3.71 (1H, d, J=14.8Hz, H-9), 3.45 (1H, m, H-10), 3.39 (1H, m, H-10), 2.98 (1H, m, H-14), 2.57 (1H, m, H-13a), 2.53 (1H, m, H-10), 2.24 (1H, m, H-12), 2.04 (1H, m, H-11), 1.93 (1H, m, H-11), 1.78 (1H, m, H-12).
13C-NMR (100MHz, CDCl
3): 125.11 (C-1), 114.83 (C-2), 157.61 (C-3), 104.55 (C-4), 103.92 (C-5), 149.42 (C-6), 148.30 (C-7), 103.02 (C-8), 53.46 (C-9), 54.91 (C-10), 21.53 (C-11), 31.03 (C-12), 60.15 (C13a), 33.03 (C-14), 55.48 (C3-OMe), 55.96 (C6-OMe), 55.90 (C7-OMe) and ring B carbon: 123.34,125.41,125.42,126.78,130.39 (overlapping carbon signals).HRMS(ESI)calcd?for[M]
+?C
23H
26NO
3363.1834，found?363.1852。
Embodiment 1:(+)-13a-(S)-deoxidation tylophorinine L-tartrate (CAT-1Tartrate) synthetic
Reactant (+)-13a-(S)-deoxidation tylophorinine 20mg and L-tartrate 8.26mg are added in the 5mL ethanol, reaction solution is heated to backflow, stirred 10 minutes, put and be chilled to room temperature, reaction solution is concentrated into 2mL, place, as seen there is the precipitation of off-white color to separate out, leach and obtain solid 22mg, mp:216～218 ℃, product is water-soluble.
Embodiment 2:(+)-13a-(S)-deoxidation tylophorinine maleate (CAT-1Maleate) synthetic
Reactant (+)-13a-(S)-deoxidation tylophorinine 50mg and toxilic acid 16mg are added in the 10mL ethanol, reaction solution is heated to backflow, stirred 10 minutes, visible solution becomes is clear and bright, puts and is chilled to room temperature, reaction solution is concentrated into 5mL, place, as seen have lurid precipitation to separate out, leach and obtain solid 45mg, mp:126～128 ℃, product is water-soluble.
Embodiment 3:(+)-13a-(S)-deoxidation tylophorinine citrate (CAT-1citrate) synthetic
Reactant (+)-13a-(S)-deoxidation tylophorinine 50mg and Citric Acid 29mg are added in the 10mL ethanol, reaction solution is heated to backflow, stirred 10 minutes, visible solution becomes is clear and bright, puts and is chilled to room temperature, reaction solution is concentrated into 5mL, place, as seen have lurid precipitation to separate out, leach and obtain solid 60mg, mp:142～144 ℃, product is water-soluble.
Embodiment 4:(+)-13a-(S)-deoxidation tylophorinine lactic acid salt (CAT-1lactate) synthetic
Reactant (+)-13a-(S)-deoxidation tylophorinine 50mg and lactic acid 14mg are added in the 10mL ethanol, reaction solution is heated to backflow, stirred 10 minutes, visible solution becomes is clear and bright, puts and is chilled to room temperature, reaction solution is concentrated into 5mL, place, as seen have lurid precipitation to separate out, leach and obtain solid 45mg, mp:115～117 ℃, product is water-soluble.
Embodiment 5:(+)-13a-(S)-deoxidation tylophorinine hydrochloride (CAT-1hydrochloride) synthetic
(+)-13a-(S)-deoxidation tylophorinine 20mg is dissolved among the methylene dichloride 5mL, under agitation feeds exsiccant HCl gas in solution, visible solution becomes faint yellow, continue logical HCl gas, react after 30 minutes the reaction solution evaporate to dryness, obtain white solid 20mg, mp:225～227 ℃.
Pharmacological testing
Experimental example 1: the mensuration of anti tumor activity in vitro (mtt assay)
In order to measure the anti tumor activity in vitro of The compounds of this invention, the compound for preparing in the embodiment of the invention to be measured, its experimental procedure is:
1. cultivate the tumour cell of normal growth, with 1 * 10
4Cell/mL is inoculated into (every hole 100 μ L) in 96 orifice plates, at 37 ℃, and 5%CO
2Cultivated 24 hours in the incubator.
2. add tested compound respectively, at 5%CO
2, cultivated 5 days in the humidity incubator fully.
3. reject nutrient solution, every hole adds 0.04%MTT 100 μ L, and similarity condition was cultivated 4 hours down.
4. the reject nutrient solution adds DMSO (every hole 150 μ L), mixes the back in mensuration wavelength 570nm, reference wavelength 450nm, and colorimetric recording light optical density, the computerized compound is to the inhibiting rate of growth of tumour cell.
Result of experiment is as shown in table 1:
The MTT The selection result of table 1 (+)-13a-(S)-deoxidation tylophorinine organic acid salt:
Annotate: A549: human lung adenocarcinoma cell; Bel-7402: human liver cancer cell; U251: human brain glioblastoma cells; BT325: human brain glioblastoma cells; A2780: Proliferation of Human Ovarian Cell; BGC-823: gastric carcinoma cells; HCT-8: human colon cancer cell.
As can be seen from the above results:
The organic acid salt of (+)-13a-(S)-deoxidation tylophorinine is the same with (+)-13a-(S)-deoxidation tylophorinine to have the remarkable vitro anti-tumor activity.
Experimental example 2:(+)-body of 13a-(S)-deoxidation tylophorinine and organic acid salt thereof in pharmacodynamic experiment
In order to measure the pharmacodynamic profile of (+)-13a-(S)-deoxidation tylophorinine and organic acid salt thereof, observed the influence of (+)-13a-(S)-deoxidation tylophorinine and 3 kinds of organic acid salts thereof to H22 tumor-bearing mice tumor growth.
1, laboratory animal
A KM kind cleaning level mouse, male, body weight 18～22g, available from Test Animal Centre, Academy of Military Medical Sciences, P.L.A, animal conformity certification number: SCXK-(army) 2007-004.Every experimental group is established 6 of animals, amounts to 10 groups, 60 animals.
2, medicine and dosage
(1), given the test agent
(+)-13a-(S)-deoxidation tylophorinine (+CAT), the tartrate (Tartrate) of (+)-13a-(S)-deoxidation tylophorinine, the maleate (Malate) of (+)-13a-(S)-deoxidation tylophorinine and the citrate (Citrate) of (+)-13a-(S)-deoxidation tylophorinine, Yu Shishan researcher seminar provides by institute of Materia Medica,Chinese Academy of Medical Sciences plant chamber; Positive control drug endoxan (CTX) is available from Beijing Friendship Hospital.
(+)-13a-(S)-deoxidation tylophorinine (+CAT) MW:363.45
Tartrate (Tartrate) MW:513.54 of (+)-13a-(S)-deoxidation tylophorinine
Maleate (Malate) MW:479.52 of (+)-13a-(S)-deoxidation tylophorinine
Citrate (Citrate) MW:573.59 of (+)-13a-(S)-deoxidation tylophorinine
(2), dosage
(+)-13a-(S)-deoxidation tylophorinine gives by high dosage 5mg/kg and low dosage 2.5mg/kg, and (tartrate Tartrate is the salt of 3 kinds (+)-13a-(S)-deoxidation tylophorinine: 7.05mg/kg and 3.53mg/kg by giving with (+)-13a-(S)-deoxidation tylophorinine of 5mg/kg and 2.5mg/kg amount after with the mole number conversion; Maleate Malate is 6.60mg/kg and 3.30mg/kg; Citrate Citrate is 7.90mg/kg and 3.95mg/kg), in H22 inoculation 24 oral administration as a child, once a day, totally 7 times.CTX abdominal injection 100mg/kg gave once in the H22 inoculation in 24 hours.
3, method
Laboratory animal entered in the SPF level environment breeding observing after 24 hours, the no abnormal experiment of being allowed for access.At the H22 knurl liquid of KM kind mouse peritoneal recovery, press 1: 3 dilution proportion with in advance with aseptic physiological saline.It is subcutaneous that diluent is inoculated in the experiment mice left fore, every injection dilution posterior tuberosity liquid 0.2mL.After all animal injections finish, be divided into 12 groups at random by requirement of experiment, 6 every group.
Begin administration in inoculation after 24 hours, 4 kinds are subjected to reagent once a day, irritate stomach.Positive control drug CTX abdominal injection, once.Write down the weight of animals every day.
4, result
The influence of table 2 (+)-13a-(S)-deoxidation tylophorinine and salt pair H22 growth of xenografted thereof
* p＜0.05, * * p＜0.01 and control group comparison
5, conclusion
As can be seen from the above results:
The organic acid salt of (+)-13a-(S)-deoxidation tylophorinine is compared quite or omits high when the high dosage medication with the prototype medicine to the inhibiting rate of laboratory animal tumor growth, inhibiting rate to the laboratory animal tumor growth when the low dosage medication is compared lower slightly with the prototype medicine, but all greater than contrast cyclome phosphamide (CTX), behind this explanation (+)-13a-(S)-deoxidation tylophorinine and the corresponding organic acid salify, its anti-tumor activity is maintained.Experimental example 3:(+)-the back blood plasma pharmacokinetics and the cerebral tissue drug distribution research experiment of 13a-(S)-deoxidation tylophorinine and organic acid salt thereof
In order to observe the pharmacokinetics feature of (+)-13a-(S)-deoxidation tylophorinine and organic acid salt thereof, blood plasma pharmacokinetics and cerebral tissue drug distribution characteristic behind (S)-deoxidation tylophorinine and the 3 kinds of organic acid salts thereof have been studied (+)-13a-.
1, experimental animal
Male ICR mouse, body weight is 20～22g, is provided by Beijing Vital River Experimental Animals Technology Co., Ltd..Animal license licensed licenser licence numbering: SCXK (capital) 2007-0001.
2, test drug
(+)-13a-(S)-deoxidation tylophorinine tartrate, (+)-13a-(S)-deoxidation tylophorinine maleate, (+)-13a-(S)-deoxidation tylophorinine citrate is provided by institute of Materia Medica,Chinese Academy of Medical Sciences an enclosure for storing grain tor seminar, is mixed with concentration 0.6mg/ml solution with distilled water during experiment.
3, test method
72 of mouse are divided 3 groups, 24 every group.Fasting 16h before the experiment freely drinks water.The oral different tylophorinine salt of mouse (6mg/kg) backs respectively at 5,15,30min, 1,2,3,4,6h get blood, centrifugal separation plasma.In addition after the oral tylophorinine salt of mouse 5,15min, 2h get cerebral tissue, add physiological saline and make 25% tissue homogenate.Tissue homogenate and plasma sample 200 μ l get supernatant liquor 5 μ l and carry out the LC/MS/MS analysis with equal-volume acetonitrile precipitation albumen.
4, test-results
(1). the blood plasma typical curve
(+)-13a-(S)-deoxidation tylophorinine with after the DMSO dissolving again with the methyl alcohol dilution, concentration is respectively 10,50,100,500,1000,2500ng/ml.In blank plasma 200 μ l, add different concns (+)-13a-(S)-deoxidation tylophorinine standardized solution 10 μ l successively, make (+)-13a-that (S)-deoxidation tylophorinine final concentration is respectively 0.5,2.5,5,25,50,125ng/ml, add 190 μ l acetonitrile precipitation albumen, get supernatant liquor 5 μ l and carry out the LC/MS/MS analysis.According to each sample collection of illustrative plates, be ordinate zou with (+)-13a-(S)-deoxidation tylophorinine peak area, its concentration (ng/ml) is X-coordinate, carries out linear regression, the results are shown in Table 3, Fig. 4.In the 0.5-125ng/ml concentration range, tylophorinine concentration and spectrogram peak area linear relationship are good in the plasma sample, and relation conefficient is 0.999.
Table 3 (+)-13a-(S)-deoxidation tylophorinine mice plasma typical curve
(2). mouse oral (+)-13a-(S)-three kinds of salt of deoxidation tylophorinine (6mg/kg) back blood plasma pharmacokinetic
Mouse oral (+)-13a-(S)-deoxidation tylophorinine tartrate, maleate and citrate (6mg/kg) back plasma drug level-time data see Table 4～6, Fig. 5.After mouse oral (+)-13a-(S)-three kinds of salt of deoxidation tylophorinine, absorb very fast, can measure the original shape medicine in the 5min blood after the administration, tartrate 15min reaches the peak, reaching peak concentration is 34.2 ± 3.1ng/ml, and maleate is that 5min reaches the peak, and reaching peak concentration is 35.4 ± 3.5ng/ml, citrate 15min reaches the peak, and reaching peak concentration is 20.1 ± 12.2ng/ml.Medicine is eliminated in the mouse body comparatively fast, and the 6h Plasma Concentration is all near lowest detectable limit after the administration.Curve during according to the medicine of mouse oral (+)-13a-(S)-deoxidation tylophorinine salt, using the DAS program adopts non-compartment model to carry out match, pharmacokinetic parameter sees Table 7, (+)-13a-(S)-three kinds of salt of deoxidation tylophorinine MRT (0-∞) in the mouse body is 1.63～2.29h, obviously is longer than prototype medicine (+)-13a-(S)-deoxidation tylophorinine (0.8h).The AUC of tartrate, maleate, citrate (0-∞) is respectively 37.85,49.91,24.56 and 33.08ug/L*h, and prototype medicine (+)-13a-(S)-deoxidation tylophorinine AUC (0-∞) is 33.67ug/L*h.The AUC of maleate (0-∞) is apparently higher than other three kinds of salt and prototype medicine (+)-13a-(S)-deoxidation tylophorinine, and AUC of tartrate and citrate (0-∞) and prototype medicine are approaching.
Table 4 mouse oral (+)-13a-(S)-deoxidation tylophorinine tartrate (6mg/kg, 11.7uM) plasma drug level
Table 5 mouse oral (+)-13a-(S)-deoxidation tylophorinine maleate (6mg/kg, 12.5uM) plasma drug level
Table 6 mouse oral (+)-13a-(S)-deoxidation tylophorinine citrate (6mg/kg, 10.5uM) plasma drug level
Table 7 mouse oral (+)-13a-(S)-deoxidation tylophorinine salt (6mg/kg) blood plasma pharmacokinetic parameters
(3). mouse oral (+)-13a-(the S)-cerebral tissue of three kinds of salt of deoxidation tylophorinine (6mg/kg) back medicine distributes and studies
Mouse oral (+)-13a-(S)-deoxidation tylophorinine tartrate, maleate and citrate (6mg/kg) hindbrain remedy,tissue substrate concentration-time data see Table 8～10, Fig. 6～8.The result shows that after mouse oral (+)-13a-(S)-deoxidation tylophorinine salt, medicine more easily enters cerebral tissue.5min after the administration can measure the original shape medicine in the cerebral tissue.The distribution peak appears in 15min after the administration, cerebral tissue.Behind the administration 2h, still can keep certain levels of drugs in the cerebral tissue.The medicament contg of tylophorinine tartrate, 3 time points of maleate all is higher than the prototype medicine, and the content of citrate then is lower than the prototype medicine.
Table 8 mouse oral (+)-13a-(S)-deoxidation tylophorinine tartrate (6mg/kg) cerebral tissue medicament contg
Table 9 mouse oral (+)-13a-(S)-deoxidation tylophorinine maleate (6mg/kg) cerebral tissue medicament contg
Table 10 mouse oral (+)-13a-(S)-deoxidation tylophorinine citrate (6mg/kg) cerebral tissue medicament contg
5, conclusion
Behind mouse oral (+)-13a-(S)-deoxidation tylophorinine tartrate, maleate and the citrate (6mg/kg), absorb very fast, peak time is 5～30min, reaching peak concentration is 18.0～35.4ng/ml, (+)-13a-(S)-three kinds of salt of deoxidation tylophorinine MRT (0-∞) in the mouse body is 1.63～2.29h, obviously is longer than the prototype medicine
(+)-13a-(S)-deoxidation tylophorinine (0.8h).The AUC of tartrate, maleate, citrate (0-∞) is respectively 37.85,49.91 and 33.08ug/L*h, and prototype medicine (+)-13a-(S)-deoxidation tylophorinine AUC (0-∞) is 33.67ug/L*h, wherein the AUC of maleate (0-∞) is apparently higher than other two kinds of salt and prototype (+)-13a-(S)-deoxidation medicine tylophorinine, and AUC of tartrate and citrate (0-∞) and prototype medicine are approaching.
Mouse oral (+)-13a-(S)-deoxidation tylophorinine tartrate, maleate and citrate (6mg/kg) back 5min can measure the original shape medicine in the cerebral tissue.The distribution peak appears in 15min after the administration, cerebral tissue.Behind the administration 2h, still can keep certain levels of drugs in the cerebral tissue.The medicament contg of (+)-13a-(S)-deoxidation tylophorinine tartrate, 3 time points of maleate all is higher than the prototype medicine, and the content of citrate is lower than the prototype medicine.
Claims (7)
1. by (+)-13a-(S)-deoxidation tylophorinine shown in the general formula (I) and different sour formed salt:
Wherein, HX represents organic acid or mineral acid.
2. according to (+)-13a-(the S)-deoxidation tylophorinine and different sour formed salt of claim 1, it is characterized in that, described salt be selected from hydrochloride, phosphoric acid salt, vitriol, hydrobromate, tartrate, citrate, maleate, lactic acid salt, salicylate, malate, benzoate, adipate, fumarate or succinate.
3. a pharmaceutical composition is characterized in that, contain effective dose as each described arbitrary compound and pharmaceutically acceptable carrier in claim 1 or 2.
4. according to the pharmaceutical composition of claim 3, it is characterized in that described pharmaceutical composition is selected from tablet, capsule, pill, injection, sustained release preparation, controlled release preparation or various particulate delivery system.
5. each described compound prevents and/or treats application in the medicine of cancer in preparation in the claim 1 or 2.
6. according to the application of claim 4, it is characterized in that described cancer is selected from human colon carcinoma, people's cancer of the stomach, human ovarian cancer, cervical cancer, liver cancer, lung cancer, carcinoma of the pancreas, lymphatic cancer and neurospongioma.
7. each described compound prevents and/or treats application in the medicine of inflammatory disease in preparation in the claim 1 or 2.
Priority Applications (5)
| Application Number | Priority Date | Filing Date | Title |
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| CN200910089238.5A CN101941967B (en) | 2009-07-09 | 2009-07-09 | Salt of 13a-(S) deoxidized tylophorinine, preparation method, pharmaceutical composition and application thereof |
| PCT/CN2010/075083 WO2011003362A1 (en) | 2009-07-09 | 2010-07-09 | Salts of 13a-(s)desoxytylophorinine, preparation methods and pharmaceutical compositions and uses thereof |
| US13/382,701 US20120190703A1 (en) | 2009-07-09 | 2010-07-09 | Salts of 13a-(s)desoxytylophorinine, preparation methods and pharmaceutical compositions and uses thereof |
| JP2012518745A JP5698741B2 (en) | 2009-07-09 | 2010-07-09 | 13a- (S) Deacidified Tyrophorinine Salt, Pharmaceutical Composition and Use |
| EP10796739.0A EP2452681B1 (en) | 2009-07-09 | 2010-07-09 | SALTS OF 13a-(S)-DEOXYTYLOPHORININE, PREPARATION METHODS AND PHARMACEUTICAL COMPOSITIONS AND USES THEREOF |
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| WO2016161538A1 (en) * | 2015-04-09 | 2016-10-13 | 中国医学科学院药物研究所 | 3-acyloxy substituted (+)-deoxytylophorinine derivatives, and preparation method, pharmaceutical composition and use thereof |
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| KR101493461B1 (en) | 2014-05-15 | 2015-02-16 | 순천향대학교 산학협력단 | Composition Comprising Deoxypergularinine and the Extract of Cynanchum atratum against tuberculosis |
| CN113292574B (en) * | 2020-02-21 | 2022-05-03 | 四川大学 | Chiral polycyclic tropane compounds, preparation method and application thereof |
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| CN101058578A (en) * | 2006-04-21 | 2007-10-24 | 中国医学科学院药物研究所 | Dextrorotation deoxidization tylophorinine, preparation method thereof, pharmaceutical composition and use thereof |
| CN101189968A (en) * | 2006-11-23 | 2008-06-04 | 南开大学 | Phenanthroindolizidine and phenanthroquinolizidine derivatives and applications of salts in pesticides |
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| CN101058578A (en) * | 2006-04-21 | 2007-10-24 | 中国医学科学院药物研究所 | Dextrorotation deoxidization tylophorinine, preparation method thereof, pharmaceutical composition and use thereof |
| CN101189968A (en) * | 2006-11-23 | 2008-06-04 | 南开大学 | Phenanthroindolizidine and phenanthroquinolizidine derivatives and applications of salts in pesticides |
Non-Patent Citations (2)
| Title |
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| CHENG-WEI YANG ET AL: "Anti-Inflammatory Mechanisms of Phenanthroindolizidine Alkaloids", 《MOLECULAR PHARMACOLOGY》 * |
| 刘湘等: "《天然产物化学》", 31 March 2005 * |
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| WO2016161538A1 (en) * | 2015-04-09 | 2016-10-13 | 中国医学科学院药物研究所 | 3-acyloxy substituted (+)-deoxytylophorinine derivatives, and preparation method, pharmaceutical composition and use thereof |
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| US20120190703A1 (en) | 2012-07-26 |
| EP2452681A4 (en) | 2012-12-19 |
| JP5698741B2 (en) | 2015-04-08 |
| JP2012532150A (en) | 2012-12-13 |
| CN101941967B (en) | 2014-08-20 |
| EP2452681B1 (en) | 2017-03-15 |
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| EP2452681A1 (en) | 2012-05-16 |
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